A switch-on gene deletion detection platform is reported based on the alteration of the effective distance between G-quadruplex DNA and silver nanoclusters (AgNCs). Moreover, the role of the G-quadruplex structure in interacting with AgNCs has also been demonstrated. The detection of the deletion mutants of LMP1 and CCR5 genes is performed as the proof-of-concept of our assay. Finally, the potential biological application of the assay was demonstrated in 10% v/v human serum.